Fluorescence lifetime imaging microscopy (FLIM) is an imaging technique that can utilize differences in the exponential decay rate of fluorescence from a fluorescent sample. A fluorophore can be excited by a photon and drop to the ground state with a certain probability based on the decay rates through a number of different decay pathways. The lifetime (decay rate) of the signal can be used to create an image, and allows for viewing of the contrast between materials with different fluorescence decay rates and materials that fluoresce at the same wavelength. Further utilizing two-photon microscopy can reduce the effect of photon scattering in thick layers of a sample, which can improve image quality. Some FLIM systems can utilize time-correlated single photon counting (TCSPC) instrumentation. Photomultiplier tubes (PMTs) and discrete time-to-digital converters (TDCs) can be used to implement TCSPC. These systems, however, can have limited speed with which FLIM images can be acquired, can be costly, large in size, and complex to implement.
Certain complementary metal-oxide-semiconductor (CMOS) processes can integrate a solid-state alternative to a PMT with timing electronics for on-chip TCSPC. These devices can be referred to as silicon photomultipliers (SiPMs) or, for TCSPC, single-photon avalanche diodes (SPADs), and can allow for arrays of detectors with improved frame rates through wide-field imaging. Detection limits for SPADs can be affected by noise, which can be in the form of the device's dark count rate (DCR). SPADs fabricated using certain processes have achieved DCRs as low as a few hundred Hz. However, certain SPADs in standard CMOS technology, which can have nodes smaller than 0.35 μm, can result in increased DCR or utilize specialized processes, such as hydrogen passivation, to reduce the DCR.
Fluorescence lifetime imaging microscopy (FLIM) can also be based on the differences in the exponential rate of decay of fluorescence from a sample. A fluorophore excited by a photon can drop to the ground state with a certain probability based on the decay rates through a number of different decay pathways. An image can then be composed using duration rather than intensity data of the signal.
Fluorescence lifetime can reveal changes in the local chemical and physical environment of a fluorophore, as well as the binding dynamics of single proteins through excited state interactions and Förster resonance energy transfer (FRET). Certain active dyes, molecular probes and even transgenic labeling strategies can utilize FRET to enable real-time observation of cellular processes both in vitro and in vivo. Certain metabolites, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), can also exhibit changes in their fluorescence lifetime during protein binding, which can be correlated with the health of the cell, with changes seen in precancerous cells and cells undergoing apoptosis and necrosis. While FRET can be detected using intensity-only measurements, quantitation can be impaired by experimental factors such as photobleaching and concentration dependent intensity fluctuations. Further, fluorescence lifetime imaging microscopy (FLIM) can be used for biological research, and can utilize time correlated single photon counting (TCSPC) instrumentation. A TCSPC detector can include photomultiplier tubes (PMTs), which can be large in size, and discrete time-to-digital converters (TDCs). FLIM microscopy can be implemented in a laser scanning configuration at a reduced complexity and cost by using only one TCSPC detector channel. However, dwell times of TCPSC channels can be on the order of 1 ms, and as such, a 128×128 pixel image can take over 16 seconds to acquire, which can prevent FLIM instrumentation from imaging real-time dynamic processes on millisecond time scales.
Accordingly, there is an opportunity for improved imaging systems.